Myeloproliferative neoplasms (MPNs) are chronic myeloid cancers that are characterized by the overproduction of mature blood cells, and that may evolve into acute myeloid leukemia. In addition to chronic myeloid leukemia with the BCR-ABL fusion gene, the three most common myeloproliferative neoplasms are essential thrombocythemia, polycythemia vera, and myelofibrosis.
Chronic myeloproliferative neoplasms (MPNs) are diagnosed and confirmed by morphologic and characteristic molecular abnormalities. Almost all cases with polycythemia vera (P-Vera) are characterized by a mutation in JAK2. Primary myelofibrosis (PMF) and essential thrombocythemia (ET) show JAK2 mutation in 40% of cases and MPL gene mutation in 2% of cases. Tests for these JAK2 mutations have greatly simplified the diagnosis of MPNs and are part of the standard screening mechanisms. However, distinguishing essential thrombocythemia with non-mutated JAK2 from the more common reactive thrombocytosis remains a diagnostic challenge.
Additional mutations have been identified in patients who have MPNs with or without JAK2 mutations, but these additional mutations affect only a small number of patients. Recently, researchers have identified the gene encoding calreticulin (CALR) as a new cancer gene that is frequently mutated in patients with MPNs. (J. Nanglia, et al., “Somatic CALR mutations in myeloproliferative neoplasms with non-mutated JAK2”, New England J. Med., Dec. 19, 2013: 369(25): 2391-2405). The recently discovered calreticulin (CALR) mutation is reported in 67% of ET cases and 88% of PMF that lack JAK2 or MPL gene mutations.
Somatic mutations in the calreticulin gene CALR are detected in peripheral blood in the ˜65-85% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients that are JAK2- and MPL-mutation negative. Molecular analysis of these three genes now allows these disease markers to be identified in >90% of MPN patients, helping to classify the disease and differentiate it from a reactive process. CALR mutation testing also has prognostic value as CALR mutations are associated with longer survival and fewer thrombotic events compared to JAK2 mutations.
Calreticulin is a calcium-binding protein involved in signaling and protein expression that is believed to be responsible for clearing misfolded proteins and involved in expression regulation. CALR mutations reported in myeloproliferative neoplasms create translation frameshifts in exon 9 which truncate the C-terminal calcium binding domain and create a novel C-terminal peptide. CALR mutations are insertions or deletions in exon 9 and highly unlikely to be missense mutation. The two most common mutations in CALR are 52-bp deletions (type 1 mutation) and 5-bp deletions (type 2 mutation). Respectively, they have been shown to account for approximately 53% and 31.7% of detected CALR mutations. Initial reports support CALR mutations as early and disease-initiating mutations that favor expansion of the megakaryocytic lineage. CALR mutations are mutually exclusive with JAK2 or MPL mutations.
Almost all mutations involve both insertions and deletions (“indel”)—some with large (>50 bp) deletion. Detecting this type of mutation with acceptable sensitivity is difficult to achieve using conventional sequencing techniques.
Allele burden has been shown to play a role in determining the clinical phenotype and disease-evolution, most likely because it reflects homozygous or hemizygous mutation. Quantification of the allele burden and the demonstration of homozygous/hemizygous mutation in MPN patients harboring the JAK2 V617F has been conducted using peripheral blood and bone marrow aspirates and cell-free plasma nucleic acid. Variation in the mutation burden of the JAK2V617F has been shown to reflect the size of the myeloproliferative clone, as well as the homozygous/heterozygous state. Homozygosity can be inferred when the percent of JAK2V617F is greater than or equal to 50%. Homozygous JAK2V617F has been observed in 25% of PV patients, while most patients with ET are heterozygous or wild-type. These findings demonstrate the impact that heterozygosity/homozygosity might have on the diverse clinical phenotypes of disease.
Disease monitoring in patients with MPNs has been utilized from bone marrow extractions, peripheral blood, as well as analyzing symptoms. While analysis of tumor samples may not be effective due to numerous constraint such as the difficulty in obtaining tumor samples that may arise, the heterogeneity between tumor localization, and the invasive approach to extract a tumor sample.
A non-invasive method for disease monitoring is through analyzing tumor-derived circulating DNA found in blood plasma. Circulating DNA in blood plasma can be found either as cell-free circulating tumor DNA (ctDNA) or in circulating tumor cells (CTCs). The current understanding is that tumors undergoing necrosis or apoptosis may deposit cell-free fragments of DNA into the bloodstream, which may correlate with prognosis and tumor staging. Common oncogenic DNA mutations have rarely been found in cell-free circulating DNA of healthy individuals, indicating that analyzing ctDNA may be specific to diseased patients.
In patients with MPNs, utilizing blood plasma rather than peripheral blood or bone marrow may be superior for the classification, prognosis, and surveillance of the various disease types. Blood plasma may be more advantageous by being less diluted with normal cell DNA, and more concentrated with tumor-specific nucleic acid, increasing the sensitivity of molecular diagnostics, while being able to be extracted less invasively. Sequencing of plasma samples have been found to be reliable in distinguishing between heterozygosity and homozygosity/hemizygosity for the JAK2 V617F mutant allele while also revealing JAK2 V617F mutants in 7% of patients who were negative by cell analysis.
Fragment length analysis (FLA) is a reliable technique in detecting this type of mutation. Furthermore, FLA allows quantifying the mutant DNA and better evaluation of tumor load. Determining tumor load can be confusing and difficult when the mutation is biallelic. Distinguishing between patients with single allele mutation from those with biallelic mutations might add another dimension in predicting clinical behavior and determining the tumor load.